Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.


Introduction
The interactions between T cells and APCs form the cornerstone of adaptive immunity.Within the intricate milieu of lymphoid tissue, T cell priming stands out as a discrete and precise process, occurring amidst the multitude of dynamic cellular interactions.Multimerized peptide-major histocompatibility complex (pMHC) labeling has been the gold standard tool for capturing antigen-specific T cells from a polyclonal repertoire [1].However, such multimers, most commonly tetramers, have limitations in monitoring dynamic interactions of antigen-specific T cells in real-time due to their blocking effect on the immune synapse.Likewise, currently available T cell receptor like (TCRL), also known as peptide-in-groove (PIG), antibodies that detect pMHC complexes are known to disrupt the immune synapse, making them unsuitable for real-time monitoring of antigen-specific T cell interactions [2,3].Although alternative approaches for labeling antigenic peptides with conventional tags such as fluorescent proteins, fluorophores, and biotin-streptavidin have been used, these tags have limitations such as size constraints, membrane impermeability, and photophysically unstable nature, which restrict their use in applications for dynamic monitoring [4][5][6][7].
To overcome these drawbacks, we have developed a highly specific and minimally disruptive labeling strategy for studying antigen-specific T cell-APC interactions.Our approach involves introducing a sixamino acid tetracysteine tag (CCPGCC) on antigenic peptides and inducing fluorescence using membrane permeable thiol-reactive Arsenical Hairpin (AsH) probes [5,8].We have engineered the OVA(323-339) peptide by placing the tetracysteine tag at either the amino or the carboxy terminus, away from the TCR and MHC binding sites, to avoid disrupting T cell-APC synapse in vitro and in vivo [9].Owing to these modifications, our labeling strategy preserves the T cell priming ability and allows for the detection of the fluorescent signal on the surface of APCs in a MHCII-dependent manner.Using flow cytometry and live microscopy, we found that the labeled peptide is readily taken up by the cognate CD4 + T cells with precise specificity in vivo, as previously shown for pMHC complexes taken up from the immune synapse via processes such as trogocytosis and transendocytosis [10][11][12][13][14]. Furthermore, we have created new variants of the tetracysteine-tagged OVA(323-339) peptide with altered TCR-binding sites and have shown that the AsH probe labeling can track the TCR mediated uptake of nuanced pMHC complexes.This indicates that our labeling strategy is highly robust and forges a novel path for investigating immune responses against altered antigens such as those derived from self, microbiota and tumor microenvironment.Overall, AsH probe mediated immune monitoring offers significant advantages over existing methods, making it a versatile tool for in vitro and in vivo applications including live imaging of the T cell synapse within intact tissue architecture.This novel approach will further our understanding of T cell-mediated immune responses by also complementing tetramers for sorting antigen-specific T cells with precision and extending it to newly discovered epitopes.

Signal intensity is determined by the sequence and placement of tetracysteine tag
We first generated OVA323-339 variants by placing a tetracysteine tag "CCPGCC" at amino or carboxy termini, either consecutively or via a flexible ε-amino-caproic acid (ε-ACA) linker to prevent endosomallysosomal enzymatic digestion to the tag.This yielded four variant peptides named as C-(carboxy terminus without linker), N-(amino terminus without linker), NACA-(amino terminus with ε-ACA) and CACA-(carboxy terminus with ε-ACA) (Fig 1a).We used these variants to load mature C57BL/6 CD11c + splenic DCs from C57BL/6 mice and induced fluorescence by treating cells with Fluorescein-AsH (FlAsH) probe.
Flow cytometry analysis showed that CACA-peptide combination but not the other tested variants induced a distinguished fluorescent signal (Fig 1b).Cysteine is naturally oxidation prone and this may reduce FlAsH binding to tetracysteine tag.We addressed this problem by pre-treating OVACACA with tris-2carboxyethylphosphine (TCEP), an irreversible reducing agent that blocks cystine disulfide bridge formation and elicited a consistent FlAsH signal (Fig. 1c).Moreover, we used another AsH probe excitable by 561 nm laser, called ReAsH, to occupy non-specific cysteine residues that would otherwise attract FlAsH [15].Pre-incubation of DCs with ReAsH improved signal to noise ratio significantly (Fig 1d).We have created additional OVA variants to enhance signal intensity.These variants include one with two tetracysteine residues and another with a longer tetracysteine tag that withstands the British anti-Lewisite (BAL) wash, a crucial step to reduce background labeling by removing excess AsH probe [16].However, new variants provided FlAsH intensity comparable to the original CACA variant, indicating no additional benefit for longer tags (Fig 1e).

Tetracysteine tag does not alter the immunogenicity of peptide
Next, we sought to compare the CD4 + T cell priming ability of four tetracysteine-tagged OVA323-339 variants and that of native peptide in vitro.To do so, naïve CD4 + T cells were isolated from OVA323-339 specific TCR transgenic OTII mice, labelled with a proliferation dye and cocultured with mature CD11c + DCs that were pre-loaded with equimolar amount peptides for three days.Proliferation and activation status of OTII T cells were not altered by the presence or placement of the tag (Fig 2a-d

AsH-probe robustly tracks pMHCII complexes
To delineate whether FlAsH labeling reports free vs. MHCII-bound peptide in cells, we initially assessed how surface MHCII expression of cells correlates with the OVACACA-FlAsH signal.C57BL/6 splenocytes were incubated with OVA-Biotin or OVACACA and peptide distributions among the T cells, B cells and DCs were quantified by flow cytometry using streptavidin or FlAsH (Fig 4a).Both streptavidin and FlAsH signals correlated significantly with MHCII expression, providing the strongest signal in DCs, where CD4 + T cells served as a negative control due to lack of MHCII expression in mouse T cells [21] (Fig. 4b, Figure 4-Figure supplement 1).It is worth mentioning that streptavidin detection of biotinylated peptide is a viable option for quantifying peptide loading on cells.However, size and membrane impermeable nature of streptavidin make it unsuitable for assays that include live monitoring of the T-DC interaction [22].Next, we addressed whether FlAsH labeling of OVACACA-pulsed DCs is dependent on the MHC expression.CD11c + DCs were isolated from MHCII single knockout or MHCI and II double knockout (KO) mice, labeled with tracking dyes and mixed 1:1 with WT C57BL/6 DCs.Subsequently, they were incubated with OVACACA and treated with ReAsH and FlAsH as in Fig. 1 (Fig 4e).To make a fair comparison between WT and KO DCs and minimize autofluorescence, we performed adoptive transfers, aiming to facilitate in vivo elimination of apoptotic and possibly defective KO DCs [23,24].Flow cytometry analysis of the draining lymph node demonstrated that WT DCs, but not MHCII KO DCs, harbored FlAsH.Two-photon microscopy of the lymph node further confirmed these findings, indicating that FlAsH robustly marks

OVACACA-MHCII complexes (Fig 4e-h).
To address how FlAsH acquisition from synapse marks antigen-specific vs. bystander T cells, DCs were labeled with ReAsH and cell membrane dye PKH-26, pulsed with OVACACA, Eα(52-68) or left unpulsed.Next, they were treated with FlAsH and injected into the recipient mice via footpad.Subsequently, 1:1 mixture of labeled naïve OTII and Eα-specific T cells were adoptively transferred into via retroorbital sinus i.v. and the popliteal lymph nodes of the recipients were removed 36-42 h after the transfer (Fig. 5a).Flow cytometry analysis showed that OTII and Eα-specific T cells both acquired PKH-26 from double-pulsed DCs, indicating that they captured DC cell membrane from synapse (Fig 5b).This process is termed as TCR-mediated trogocytosis and has previously been described as a feature of the DC-T cell synapse [11].

Tetracysteine-tagging of peptides provides a sensitive tool to visualize interactions with altered TCR affinity
The lack of adaptable antigen-targeted probes presents a significant obstacle in the dynamic visualization of antigen-specific immunity.To address this gap, we sought to determine robustness of tetracysteine tagging in visualizing nuanced pMHCII variants.We created three variants by introducing alanine mutations at the TCR binding residues 331, 333, and 335 of OVACACA (Fig 6a).The mutations at 331 and 333, depicted as variants 1 and 2 respectively, were previously reported to abolish the binding of OTII TCR to OVA323-339 [9].Although the residue 335 is flanked between the core OTII TCR binding residues 333 and 336, mutant 335 was shown to elicit normal proliferative response in vitro [9].We addressed how pMHCII uptake by T cells is influenced by the mutation of TCR binding residues of OVACACA.DCs were isolated from CD45.1 congenic mice, loaded with variant peptides and cocultured with naïve CD45.2 OTII T cells for three days (Fig 6b-e).In line with earlier observations, variants 1 and 2 interrupted T cell priming while variant 3 led to a proliferative response similar to native peptide (Fig 6c-d).TCR mediated uptake of the FlAsH-labeled pMHCII complexes followed the same pattern and only variant 3 complexes produced a comparable signal to that of OVACACA (Fig 6e).These suggest that AsH-probe labeling can be a sensitive method to track low affinity/ nuanced pMHCII complexes.

Discussion
Dynamic monitoring of antigen-specific T cell responses in vivo poses challenges due to the limited availability of reagents and the size constraints of the immune synapse.Here, we address this issue by presenting a novel strategy for visualizing T cell-APC interactions via tetracysteine tagging of antigenic peptides.While tetracysteine tagging has been previously applied in cell biology and virology, these applications have been mostly limited to cell lines and in vitro studies in immunology unrelated experimental settings [25][26][27][28].On the other hand, our technique labels antigenic peptides and tracks pMHCII complexes in primary APCs during their interactions with T cells both in vitro and in vivo.Our approach is superior as compared to other strategies for monitoring T cell-DC interactions, in the way it robustly targets specific pMHCII complexes in real time in vivo.There is no need for special organisms or genetic modifications, and the technique can easily be expanded to new antigens and T cell-APC pairs from both mice and humans.This strategy can, for instance, be integrated into validation efforts for antigen discovery and utilized in the investigation of the synapses of newly deorphanized human T cells, a largely unexplored area where tools are scarce [29][30][31][32].Given that is a two-part system (tag and FlAsH), it also carries the added capability of being inducible at different time points to allow for increased flexibility in interrogating pMHCII-TCR interactions over time.On the other hand, this introduces a high background due to the non-specific binding of FlAsH to endogenous cysteines.Thus, we implemented a ReAsH pretreatment strategy, successfully improving the signal-to-noise profile for FlAsH.It's worth mentioning our unpublished attempts to load dendritic cells (DCs) with preconjugated peptide and FlAsH to mitigate the background.Unfortunately, this endeavor led to almost a complete loss of the FlAsH signal and has not been pursued further.Therefore, exploring new peptide probes chemically conjugated to small organic dyes emerges as a potential avenue for future investigation.
Most important of all, our experiments demonstrate that tetracysteine-tagged peptides can be transferred from DCs to T cells via synaptic processes, such as antigen-specific pMHCII trogocytosis/transendocytosis, marking acceptor T cells for several days.Although the turnover of pMHCII in acceptor T cells and its temporal relationship to the TCR downstream events require further elucidation, our visualization of tetracysteine-tagged pMHCII can complement tetramers in detecting antigen-specific T cells in vivo.
Additionally, we show that tetracysteine tagging of pMHCII does not broadly alter or disturb T cell-DC synapse by functional read-outs.Although the ratio of peptide-FlAsH conjugates to free peptide in DCs would be needed to support this claim, our approach offers an advantage over tetramers in that it is suitable for real-time monitoring of the immune synapse.Finally, it is worth noting that tetracysteine tags can be visualized as electron-dense particles by Transmission Electron Microscopy [33,34], providing potential for the detailed investigation of T-DC synapse and subcellular localization of pMHCII complexes.
Although TEM application was out of scope for this study, it offers a promising avenue for future research.
In conclusion, the tetracysteine labeling approach described here provides a powerful and unique tool to advance our understanding of T cell-APC interactions, offering new opportunities to investigate the immune response and develop improved immunotherapies.
Cell cultures were maintained in sterile complete Roswell Park Memorial Institute (RPMI) media.The RPMI 1640 medium was supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 µM streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 µM 2-mercaptoethanol, and 10 mM HEPES (Thermo Fisher Scientific).For magnetic separations, cells were maintained in filtered and degassed magnetic-activated cell sorting (MACS) buffer.The MACS buffer consisted of PBS (Lonza) supplemented with 0.5% BSA (Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich).For splenic dendritic cell (DC) isolation, Liberase TM (Blendzyme) and DNase I were purchased from Roche.50 mg Liberase was dissolved in complete RPMI, resulting in a final concentration of 5 mg/mL.The solution was kept on ice, stirred gently every 5 minutes for 30 minutes and aliquoted into 1 mL portions.10 mg/mL DNAse stock solution was prepared with 0.15 M NaCl and divided into 500 µL aliquots.Both enzymes were stored at -20ºC.For AsH probe labeling, peptides were pre-treated with fresh Tris Carboxy Ethyl Phosphene (TCEP, Sigma-Aldrich) to protect tetracysteine residues from oxidation.10 mM ReAsH stock solution was prepared by adding 45 µL sterile cell-culture grade DMSO (Sigma-Aldrich) to 250 µg solid ReAsH (Cayman Chemical) and 10 mM FlAsH solution was prepared by adding 150uL DMSO to 1 mg FlAsH powder (Cayman Chemical).British Anti-Lewisite (BAL, Alfa Aesar) and HBSS with Ca ++ and Mg ++ (Thermo Fisher Scientific) was used to wash excess FlAsH off the cells.BAL is resuspended in sterile degassed water to obtain 8 mM stock solution.FlAsH, ReAsH and BAL are highly sensitive to oxidation, therefore their activity may decline in subsequent uses.This may be averted, at least in part, by quickly purging stock solution containers with 4M Argon gas (Sigma-Aldrich, 501247), placing container in a 50mL tube, purging again with Argon before storing FlAsH and ReAsH at -20ºC, BAL at 4ºC.We do not recommend storage longer than one month or repeated use of the same reagent stock.

DC isolation, peptide loading and -AsH probe labeling
Mature splenic DCs were isolated as previously described [35] and resuspended in complete RPMI.
Peptides were added into 20 mM TCEP solution at twice the desired concentration for loading DCs.The solution was vortexed thoroughly and incubated at 37ºC for 30 minutes.Meanwhile, ReAsH stock solution was diluted to 100 uM in complete RPMI and added on DC suspension to obtain 1 uM final ReAsH concentration.Cells were incubated at 37ºC for 30 minutes and washed twice with complete RPMI.ReAsH labeled DCs were mixed with equal volume of peptide-TCEP solution, incubated at 37ºC for 30 minutes, washed twice and resuspended in complete RPMI to proceed with FlAsH labeling.Flash was added to cell suspension to achieve 1 uM final concentration, incubated at 37ºC for 30 minutes and washed twice with complete RPMI.To remove excess FlAsH and reduce non-specific binding, cells were resuspended in 240 uM BAL diluted in HBSS with Ca ++ and Mg ++ , washed twice, then washed a third time with PBS and resuspended in PBS.

DC-T cell cocultures
Freshly isolated DCs were pulsed with peptide in complete RPMI and were incubated at 37 °C for 30 min followed by three washes with complete RPMI to remove unbound peptide.Pulsed DCs were then treated with -AsH as decribed above then seeded into flat-bottom 96-well plates (Corning) at a density of 5 × 104 to 1 × 105 cells per well.T cells and iTreg cells were labeled with e450 or e670 (eBioscience) according to the manufacturer's protocol.Cells were cocultured at 37°C for for indicated periods, at a 1:10 ratio of DCs to T cells for proliferation assays and 1:1 for trogocytosis assays.

Trogocytosis assay
Trogocytosis assays were perfomed as described in [20].Briefly, freshly isolated splenic DCs were labeled with 4μM PKH26 (Sigma-Aldrich) for labeling of the cell membrane as described in Puaux et al.48.DCs were then pulsed with 10 μM peptide, treated with -AsH and were cocultured with T cells for up to 18 h.Cell conjugates were dissociated by washing cells with MACS buffer with 2 mM EDTA; cell suspensions were prepared for flow cytometry.

Flow cytometry
Cells were stained in FACS buffer containing fluorochrome-conjugated antibodies for 30 minutes at 4°C, protected from light.Data acquisition was performed with BD LSRFortessa and BD LSR II cytometers (BD Biosciences).Data analysis was performed with FlowJo v10.8.1.

Adoptive transfer
Mice were anesthetized in an induction chamber with 1.5% isoflurane USP (Baxter).Pulsed DCs were adoptively transferred through the right footpad in 50uL sterile PBS.T cells labelled with e450 or e670 (Thermo Fisher Scientific) were injected intravenously in 100uL sterile PBS through the retro-orbital sinus.

Confocal microscopy
35mm dishes with a 14mm glass coverslip bottom (MatTek) were pre-treated with 10ug/ml fibronectin (Sigma-Aldrich) in PBS for 1h at room temperature and washed twice.Splenic DCs were added onto the glass coverslip in complete RPMI and pulsed with 5-10 μM peptide at 37°C for 1 hour.Excess peptide was washed off with complete RPMI before addition of CD4 T cells.Co-cultures were imaged for 3-18h using a Leica SP-8 inverted microscope (Leica Microsystems) equipped with a full range of visible lasers, two hybrid detectors, three photomultiplier detectors, and a motorized stage.63× objective (Leica Microsystems) was used and microscope configuration was set up for 3D analysis (x, y, z) of the cellular layer.The following lasers were used: diode laser for 405 nm excitation; argon laser for 488 and 514 nm excitation; diode-pumped solid-state laser for 561 nm; and HeNe laser for 594 and 633 nm excitation.All lasers were tuned to minimal power (between 0.3% and 2%) to prevent photobleaching.z stacking of images of 10-12 µm were collected.Mosaic images of large cell culture areas (1 mm2) were generated by acquiring multiple z stacks using the Tile scan mode and were assembled into tiled images using LAS X v4.0 (Leica Microsystems).

Intravital two-photon laser-scanning microscopy of mouse popliteal lymph nodes
Mice were anesthetized in 1.5% isoflurane USP (Baxter) through a rodent nose cone followed by surgery to expose the right popliteal lymph node.Warm PBS was added to maintain lymph node moisture.Mouse body temperature was maintained with an infrared blanket (Braintree Scientific) throughout the imaging process after which they euthanized through cervical dislocation under anesthesia.Mice were imaged on a glass stage with a two-photon laser-scanning microscopy setup with a Leica SP8 inverted confocal microscope with dual multi-photon lasers, Mai Tai and InSight DS (Spectra-Physics), L 25.0 waterimmersion objective, 0.95 NA (Leica Microsystems), and a 37 °C incubation chamber (NIH, Division of Scientific Equipment and Instrumentation Services).The Mai Tai laser was tuned to 890 nm to excite e450and FlAsH; the InSight DS laser was tuned to 1,150 nm to excite DsRed/ PKH-26/ ReAsH and e670.
For time-lapse imaging, a z stack consisting of 10-12 single planes (5 μm each over a total tissue depth of 50-60 μm) was acquired at 15 second intervals for a total period of 1-4 h.

Two-photon laser-scanning microscopy of live lymph node sections
Live lymph node sections were imaged ex vivo by two-photon microscopy.To maintain tissue architecture, popliteal lymph nodes were immediately transferred to PBS with 1% BSA and kept on ice immediately following harvest.Residual fat and connective tissue were removed using a Leica MZ6 StereoZoom microscope (Leica Microsystems).Lymph nodes were suspended in 38 °C agarose in DMEM and kept over ice.Following complete agarose solidification, complete RPMI was added and blocks were cut containing one lymph node each.Lymph node blocks were cut into 200 µm sections with a Leica VT1000 S vibrating blade microtome (Leica Microsystems) at speed 5 in ice-cold PBS and frozen at -80 °C.Prior to imaging, complete RPMI medium was added to tissue sections and incubated at 37 °C for 2 h.Sections were held down with tissue anchors (Warner Instruments) in 35mm dishes with a 14mm glass coverslip bottom (MatTek) and were imaged with a Leica SP8 inverted five-channel confocal microscope equipped with an environmental chamber (NIH, Division of Scientific Equipment and Instrumentation Services) and a motorized stage.The microscope configuration was set up for fourdimensional analysis (x, y, z, t) of cell segregation and migration within tissue sections.A diode laser for 405 nm, an argon laser for 488 and 514 nm, a diode-pumped solid-state laser for 561 nm, and a HeNe laser for 594 and 633 nm excitation wavelengths were tuned to minimal power (between 0.3% and 2%).
z-stack of images of 10-25 µm were collected.Mosaic images of whole lymph nodes were generated by acquiring multiple z stacks using a motorized stage to cover the whole lymph node area and assembled into a tiled image with the LAS X software.For time-lapse analysis of cell migration, tiled z stacks were collected over time (1-4 h).

Image analysis
Analysis of confocal and intravital two-photon images was performed using Imaris v9.2.1 and v10.0.0 (Bitplane) as previously described [35].In brief, e450 and e670 signals were utilized for reconstructing the 3D structure of T cells and iTreg cells as surface objects.To reconstruct the 3D structure of DCs, overlapping DsRed, PKH-26, and ReAsH signals were employed.Imaris v10.0.0's built-in algorithms were used to calculate 3D volumes, track velocities, surface-to-surface colocalization parameters, and FlAsH (green) intensities of cells.cells) and e670-labeled polyclonal T cells (1 × 10 6 cells) were injected i.v.Popliteal lymph nodes were either imaged at 18 h or analyzed by flow cytometry at 18 h or 48 h post-transfer.a, Experimental approach.b, Flow cytometry plots gating on DsRed+ DCs over 48h (top panel) and demonstrating the OVACACA levels (FlAsH, bottom panel).c, Gating strategy for T cells of the recipient (CD45.1)and donor (CD45.2). d, T cell levels of OVACACA (FlAsH), MHCII, and OVA-biotin (detected with streptavidin) at 48 h following adoptive transfer.a-d, Data are representative of three independent experiments with n=3 mice per time point.e-i, Summary data from live microscopy showing OTII cell blasting (e), DC-T cell interaction with 45 min intervals (f), plots for the DC-T cell contact duration and volume, graphs for the FlAsH intensity limited to the DC-T cell contact regions (g-h) and average Flash intensity acquired by T cells (i).Data are representative of four independent experiments with n=2 mice per experiment.P values were calculated using two-sided Welch's t-test.

Simple linear regression and
Pearson correlation and were used to demonstrate the relationship between surface MHCII and peptide load, gray area and dotted lines mark the standard error of the fitted line (b,c).d, Schematic depicting in vivo adoptive transfer approach.WT and KO DCs were labeled, mixed, pulsed with 5 μM OVACACA, labeled with FlAsH and adoptively transferred to WT recipients via footpad (1-2 × 10 6 cells).e-f, Flow cytometry plots (e) and graphs (f) showing FlAsH intensity of WT, MHCII-KO, or MHCI/II double-KO DCs that had migrated to the lymph node within 18 h following adoptive transfer.Data are representative of two independent experiments with n=4 mice per group.P values were calculated using two-sided Student's t-test.g-h, Microscopy images of popliteal lymph node following anti-CD31 (red) antibody injection i.v., yellow arrows point to WT, white arrows point to KO DCs (g), FlAsH intensity of adoptively transferred DCs (h).Data are representative of four independent experiments with n=2 mice per experiment.P values were calculated using two-sided Welch's t-test.Video 2: ReAsH-labeled DCs were double-pulsed with OVACACA and LCMV GP61-80, treated with FlAsH (green) and adoptively transferred via footpad.Naïve e450-labeled OTII and e670-labeled SMARTA T cells were mixed 1:1 and injected i.v.via retroorbital sinus.Popliteal lymph nodes of the recipient mice were surgically removed 18 h after transfer, sectioned and recovered at 37 °C until the resumption of cell migration in the tissue.Sections were then imaged at 37 °C by confocal microscopy for 4 h.Video shows that both OTII (blue) and SMARTA T cells (magenta) have sustained contacts with the DC (Red).Gray: 3D surface rendering for the DCs.Data are representative of three independent experiments.6 biological replicates (one per symbol).Simple linear regression and Pearson correlation and were used to demonstrate the relationship between surface MHCII and peptide load, gray area and dotted lines mark the standard error of the tted line (b,c).d, Schematic depicting in vivo adoptive transfer approach.WT and KO DCs were labeled, mixed, pulsed with 5 μM OVA CACA , labeled with FlAsH and adoptively transferred to WT recipients via footpad (1-2 × 10 6 cells).e-f, Flow cytometry plots (e) and graphs (f) showing FlAsH intensity of WT, MHCII-KO, or MHCI/II double-KO DCs that had migrated to the lymph node within 18 h following adoptive transfer.Data are representative of two independent experiments with n=4 mice per group.P values were calculated using two-sided Student's t-test.g-h, Microscopy images of popliteal lymph node following anti-CD31 (red) antibody injection i.v., yellow arrows point to WT, white arrows point to KO DCs (g), FlAsH intensity of adoptively transferred DCs (h).Data are representative of four independent experiments with n=2 mice per experiment.P values were calculated using two-sided Welch's t-test.

Supplementary Files
This is a list of supplementary les associated with this preprint.Click to download.
Although both T cell types trogocytosed double-pulsed DC cell membrane, FlAsH signal was picked up only by OTII T cells, suggesting that FlAsH tracks pMHCII mobility with a high precision (Fig 5c).Furthermore, we visualized dynamic CD4 + T cell-DC contact and pMHCII mobility in vivo at 18 h after the transfer.DCs were labeled with ReAsH and pulsed with OVACACA and/or LCMV GP(61-80) and treated with FlAsH.Recipient mice were injected with unpulsed, OVACACA single or OVACACA-GP(61-80) double pulsed DCs via footpad along with a 1:1 mixture of labeled naïve OTII and SMARTA T cells i.v.via retroorbital sinus.Popliteal lymph node microscopy confirmed that OTII and SMARTA T cells had stable contacts with double-pulsed DCs (Fig 5d, Video 2).Similar to Eα-specific T cells, SMARTA also failed to pick up FlAsH signal despite showing signs of antigen-specific contact such as increased volume, reduced sphericity and decreased track speed (Fig 5 e-f).
Prism v9.5.1 (GraphPad).Statistical tests and relevant P values for each figure are indicated in the figure or figure legend.

Figure 1 .
Figure 1.FlAsH labeling elicits fluorescence depending on the location of the tetracysteine tag.a, AsH-probe labeling strategy for the tetracysteine-tagged peptides and the four OVA323-339 variants generated for the study.b, FlAsH signal intensity on splenic DCs pulsed with tagged OVA323-339 variants.c, FlAsH signal intensity on splenic DCs pulsed with CACA-variant following treatment with TCEP, a strong reducing agent.d, Signal intensity following pre-treatment of splenic DCs with ReAsH, followed by pulsing with CACA-variant and FlAsH treatment.e, FlAsH signal intensity of modified CACA-variant peptide with a duplicated CCPGCC sequence (CACA-2xTetracysteine) and CACA-variant peptide flanked with BAL-resistant peptide sequences (CACA-BAL resistant).Data in b-d are representative of three independent experiments, each performed with three biological replicates.

Figure 2 .
Figure 2. Tetracysteine tag does not alter the T cell priming ability of the native peptide.a-d, DCs were loaded with the indicated peptides (5 μM) and co-cultured with e450-labeled naïve OT-II T cells (5 × 10 4 ) at a 1:10 DC to T cell ratio.a, Histograms (a) and the bar graph (b) demonstrate the proliferation status of T cells at 72 hours.Histograms (c) and the bar graph (d) depict the surface expression of CD44, a T cell activation marker.The bars (b,d) indicate the mean of three biological replicates; the error bars show the standard deviation.Data are representative of three independent experiments.P values refer to comparisons between each group and the native peptide, calculated using one-way ANOVA with Dunnett's multiple comparisons test.e-g, Peptide-pulsed DCs (2 × 10 5 ) were treated with FlAsH, cocultured 1:1 with e450-labeled naïve OT-II T cells and imaged for real-time interactions for 3 h (blue, OTII; green, FlAsH).e, Time lapse images depict the clustering of OTII T cells around OVACACA-pulsed DCs.f, Histocytometry plots for OTII T cell volume and sphericity.g, Graph shows the average track speed of OTII T cells.The lines mark the means of n = 3 replicates (one per symbol).Data are representative of two independent experiments.P values were calculated using a one-way ANOVA with Tukey's test.

Figure 3 .
Figure 3. T cells acquire tetracysteine-tagged peptide from immune synapse.DsRed + splenic DCs were double-pulsed with 5 μM OVACACA and 5 μM OVA-biotin and adoptively transferred into CD45.1 recipients (2 × 10 6 cells) via footpad.Naïve e450-labeled OTII (1 × 10 6 cells) and e670-labeled polyclonal T cells (1 × 10 6 cells) were injected i.v.Popliteal lymph nodes were either imaged at 18 h or analyzed by flow cytometry at 18 h or 48 h post-transfer.a, Experimental approach.b, Flow cytometry plots gating on DsRed+ DCs over 48h (top panel) and demonstrating the OVACACA levels (FlAsH, bottom panel).c, Gating strategy for T cells of the recipient (CD45.1)and donor (CD45.2). d, T cell levels of OVACACA (FlAsH), MHCII, and OVA-biotin (detected with streptavidin) at 48 h following adoptive transfer.a-d, Data are representative of three independent experiments with n=3 mice per time point.e-i, Summary data from live microscopy showing OTII cell blasting (e), DC-T cell interaction with 45 min intervals (f), plots for the DC-T cell contact duration and volume, graphs for the FlAsH intensity limited to the DC-T cell contact regions (g-h) and average Flash intensity acquired by T cells (i).Data are representative of four independent experiments with n=2 mice per experiment.P values were calculated using two-sided Welch's t-test.

Figure 4 .
Figure 4. AsH probe reports the peptide loaded on the MHCII.a-c, Relationship between surface MHCII expression and peptide load.a, Experimental scheme.b, Plots (top) show the surface levels of MHCII and OVA-biotin, graph (bottom) shows their correlation.Data are representative of two independent experiments performed with n = 2 biological replicates (one per symbol).c, Surface levels of MHCII and OVACACA (FlAsH) and their correlation.Data are representative of two independent experiments performed with n = 6 biological replicates (one per symbol).Simple linear regression and Pearson correlation and were used to demonstrate the relationship between surface MHCII and peptide

Figure 5 .
Figure 5. FlAsH-pMHCII is incorporated by cognate T cells in an antigen-specific manner.a-c, DCs were labeled with ReAsH and PKH-26, single/ double-pulsed with 5 μM OVACACA, Eα(52-68) or left unpulsed.They were labeled with FlAsH and adoptively transferred into WT mice (0.5-1 × 10 6 cells) via footpad.Naïve e450-labeled OTII and e670-labeled Eα-specific T cells were mixed 1:1 (0.25-0.5 × 10 6 / T cell type) and injected i.v.Popliteal lymph node was analyzed by flow cytometry at 36-42 h post-transfer.a, Experimental scheme.b, Summary plots.c, PKH-26 and FlAsH MFI in OTII and Eα-specific T cells.Data are representative of two independent experiments with n=3 mice per group.P values were calculated using two-way ANOVA with Sidak's multiple comparisons test.d-f, DCs were labeled with ReAsH and single/ double-pulsed with 5 μM OVACACA, LCMV GP61-80, labeled with FlAsH and adoptively transferred into WT mice (1-2 × 10 6 cells) via footpad.Naïve e450-labeled OTII and e670labeled SMARTA T cells were mixed 1:1 (0.5-1 × 10 6 / T cell type) and injected i.v.Live popliteal lymph node sections were imaged at 18 h.d, Time series representative of DC-T cell interactions.e, Average Flash intensity of the adoptively transferred cells.f, T cell blasting (left and middle panels) and motility (right) following adoptive transfer with DCs pulsed with the indicated peptides.Data are representative of three independent experiments with n=2 mice per group.P values were calculated using one-way ANOVA with Tukey's multiple comparisons test.

Figure 6 .Supplementary File 1 :
Figure 6.Nuanced TCR-pMHCII interactions can be discerned by FlAsH labeling of peptides.a-b, Graphical depiction of the alanine-mutants for OVACACA TCR binding site and experimental approach.0,5 × 10 5 CD45.1 + DCs were pulsed with the indicated peptides, labeled with FlAsH and cocultured with 2,5 × 10 5 e450-labeled OTII cells for 3 days.c, Gating strategy for T cells and DCs.d, Representative flow cytometry plots for OTII T cell proliferation with a partial agonist (top) and two antagonist peptides (bottom).e, FlAsH signal intensity of DCs and OTII cells with partial agonist (top) and antagonist peptides (bottom) adoptive transfer with the indicated peptides followed by FlAsH treatment.Data are representative of two independent experiments performed with n = 3 biological replicates.Supplementary File 1:The sequences and molecular weights of the peptides used in the study.

Figure 4 -Video 1 :
Figure 4-Figure supplement 1: Sequential gating strategy for splenocytes to analyze CD4+ T cells, B cells and DCs.Video 1: OVACACA-pulsed DsRed + DCs were adoptively transferred via footpad.Naïve e450-labeled OTII and e670-labeled polyclonal T cells (1 × 10 6 cells) were injected i.v.via retroorbital sinus.Popliteal lymph nodes were imaged by intravital two photon microscopy at 18 h after adoptive transfer.Video shows movement and interaction of naive OT-II (blue) cells with DCs (red).OVACACA-FlAsH is visualized as cyan-green speckles in DCs and OTII cells.Polyclonal T cells (magenta) don't engage in sustained interactions with DCs and don't acquire FlAsH.Data are representative of four independent experiments.

Figures Figure 1 FlAsH
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Figure 5 FlAsH
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